The tick-borne flaviviruses are endemic throughout most of the northern hemisphere, and except for Langat, cause disease of varying severity that can have a mortality as high as 20 to 30%. Tick-borne encephalitis remains a pressing public health problem in Eastern Europe and Russia. Approximately three decades ago, these properties of antigenic cross reactivity and virulence polymorphism suggested that successful immunization might be achieved using a live, naturally attenuated tick- borne flavivirus. The impetus for this approach was the recovery of a virus from ticks in Malaysia, namely Langat virus (LGT) that did not appear to be associated with human disease under natural conditions. Before evaluating the more attenuated E5 mutant of LGT as a possible candidate for use in prophylaxis of severe human disease caused by certain members of tick-borne flavivirus group, we sought to reduce or ablate the last vestiges of virulence of E5 for mice by using a strategy that had been used successfully to reduce neurovirulence and abolish neurovasiveness of TBEV, namely substitution of structural protein genes of the tick-borne flavivirus for the corresponding genes of dengue type 4 virus (DEN4). In studies that applied this strategy to LGT, only chimeras containing the premembrane (preM) and envelope (E) structural proteins of LGT TP21 or E5 were viable. Each of the infectious LGT(preM-E)/DEN4 chimeras was at least 5,500 times less neurovirulent than its parental LGT virus in suckling mice. Significantly, the chimeras lacked detectable evidence of neuroinvasiveness after intraperitoneal (IP) inoculation of Swiss mice with 100 000 plaque-forming units (PFU), or IP inoculation of SCID mice with 10 000 000 PFU. The implications of these observations for development of a live attenuated TBEV vaccine are encouraging. The mice that twice inoculated with TP21(preM-E)/DEN4 chimera were protected completely against subsequent challenge with 100 lethal dose of the highly neurovirulent Far Eastern strain of TBEV. Based on our observations, these chimeric viruses, as well as more attenuated mutants derived from them, appear to represent promising candidate TBEV vaccine strains.